14-3-3 proteins are a family of conserved intracellular regulatory molecules that are ubiquitously expressed in eukaryotes. 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. Indeed, more than 100 signaling proteins have been reported as 14-3-3 ligands. 14-3-3 proteins may be considered evolved members of the Tetratrico Peptide Repeat superfamily. They generally have 9 or 10 alpha helices, and usually form homo- and/or hetero-dimer interactions along their amino-termini helices. These proteins contain a number of known domains, including regions for divalent cation interaction, phosphorylation & acetylation, and proteolytic cleavage, among others. There are seven distinct genetically encoded isoforms of the 14-3-3 proteins that are known to be expressed in mammals, with each isoform comprising between 242-255 amino acids. The seven 14-3-3 protein isoforms are designated as 14-3-3α/β (alpha/beta), 14-3-3δ/ξ (delta/zeta), 14-3-3ε (epsilon), 14-3-3γ (gamma), 14-3-3η (eta), 14-3-3τ/θ (tau/theta), and 14-3-3σ (sigma/stratifin).
14-3-3 proteins have a high degree of sequence similarity. Consequently, anti-14-3-3 antibodies typically recognize more than one 14-3-3 protein isoform. Several anti-14-3-3 antibody preparations that have been characterized are commercially available. For example, rabbit polyclonal antibodies that recognize 14-3-3 protein are available from Biomol, Santa Cruz Biotechnology, Upstate Biotechnology, and Assay Designs. These polyclonal antibody preparations recognize 14-3-3 eta in some form; however none are selective for the eta isoform over other 14-3-3 protein isoforms. See also Martin, H. et al., (1993) Antibodies against the major brain isoforms of 14-3-3 protein. FEBS 331:296-303. See also WO 2007/128132 filed 9 May 2007. In addition to lacking isoform selectivity, few 14-3-3 antibodies have been shown to recognize 14-3-3 protein in its native configuration.
14-3-3 proteins have been implicated in a variety of conditions. However, the ubiquity and functional diversity of 14-3-3 proteins largely precludes therapeutic application of antibodies that bind to multiple 14-3-3 protein isoforms (“pan 14-3-3 antibodies”) and/or are incapable of recognizing 14-3-3 protein in its native configuration. Moreover, particular 14-3-3 isoforms are implicated in particular conditions, which pan 14-3-3 antibodies may not confidently detect in diagnostic assays and which may not be treatable in a targeted manner by such pan 14-3-3 antibodies. For example, 14-3-3 eta and 14-3-3 gamma have been implicated in arthritis. See WO 2007/128132 filed 9 May 2007. See also Kilani et al. (2007, J. Rheum. 34: 1650-1657; WO 2007/128132) who have reported that two members of the 14-3-3 protein family, particularly 14-3-3 eta and 14-3-3 gamma, are present within the synovial fluid and serum of patients with arthritis, and these isoforms are directly correlated with the levels of MMP-1 and MMP-3 in the synovial fluid and serum.